16S from soil sample near Reboudia pinata litter time 245
Microbial genomic DNA analysis. Genomic DNA from soil in the vicinity of the litter bags, as well as from soil between trenches, was extracted from each sample using the GenaAll ExgeneTM soil DNA Mini-Isolation Kit, according to the manufacturer's instructions (GeneAll, Seoul, Korea). Partial sequences of the bacterial 16s ribosomal RNA (rRNA) gene and the fungal ITS1 region from the extracted DNA were amplified by PCR. The primers used for the 16s rRNA gene were 27F [5'-AGAGTTTGATCCTGGCTCAG-3'] and 1100R [5'-GGGTTGCGCTCGTTG-3'] (Lane, 1991). For amplification of the ITS region, the primers used were ITS1F [5'-TCCGTAGGTGAACCTGCGG-3'] and ITS2R [5'-GCTGCGTTCTTCATCGATGC-3'] (White et al., 1990). The PCR conditions used were 3 m at 98C, 20 cycles of 30 s at 98C, 30 s at 55C, and 60 s at 72C, followed by 5 m at 72C. Amplification was carried out using a Verity Thermocycler (Applied Biosystems, Thermo Fisher Scientific). The PCR products derived from amplification of specific 16s rRNA and ITS regions were purified by electrophoretic separation on a 1.5% agarose gel, followed by a further purification step involving the Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. The purified amplicons were sequenced using the Ion Torrent system.